Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media

  • Thomas J. Caperna
  • Le Ann Blomberg
  • Wesley M. Garrett
  • Neil C. Talbot
Article

DOI: 10.1007/s11626-010-9382-3

Cite this article as:
Caperna, T.J., Blomberg, L.A., Garrett, W.M. et al. In Vitro Cell.Dev.Biol.-Animal (2011) 47: 218. doi:10.1007/s11626-010-9382-3

Abstract

A serum-free, feeder cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1-wk-old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder cell layers of mitotically blocked mouse fibroblasts. In serum-free medium containing 1% DMSO and 1 μM dexamethasone, confluent monolayers of hepatocytes formed and could be maintained for several wk. Light and electron microscopic analysis showed hepatocytes with in vivo-like morphology, and many hepatocytes were sandwiched between the feeder cells. When isolated liver cells were cultured in medium without dexamethasone but with 0.5% DMSO, monolayers of cholangioctyes formed that subsequently self-organized into networks of multicellular ductal structures, and whose cells had monocilia projecting into the lumen of the duct. Gamma-glutamyl transpeptidase (GGT) was expressed by the cholangiocytes at their apical membranes, i.e., at the inner surface of the ducts. Cellular GGT activity increased concomitantly with the development of ductal structures. Cytochrome P-450 was determined in microsomes following addition of metyrapone to the cultures. In vivo-like levels of P-450s were found in hepatocyte monolayers while levels of P-450 were markedly reduced in cholangiocyte monolayers. Serum protein secretion in conditioned media was analyzed by Western blot and indicated that albumin, transferrin, and haptoglobin levels were maintained in hepatocytes while albumin and haptoglobin declined over time in cholangiocytes. Quantitative RT-PCR analysis showed that serum protein mRNA levels were significantly elevated in the hepatocytes monolayers in comparison to the bile ductule-containing monolayers. Further, mRNAs specific to cholangiocyte differentiation and function were significantly elevated in bile ductule monolayers in comparison to hepatocyte monolayers. The results demonstrate an in vitro model for the study of either porcine hepatocytes or cholangiocytes with in vivo-like morphology and function.

Keywords

Cell cultureCholangiocyteDuctHepatocyteP-450γ-Glutamyl transpeptidaseSerum-free

Supplementary material

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Supplementary Fig. S1

Effects of somatostatin or secretin on the fluid transport of bilary canaliculi and bile ductules of porcine hepatocytes cultured in DEX/DMSO or 0.5% DMSO medium, respectively. Before addition of somatostatin or secretin to the culture medium of 0.5% DMSO-induced bile ductules (a) or DEX/DMSO-induced hepatocytes with biliary canaliculi (b). Note arrows indicating lumen of bile ductule or biliary canaliculi between individual hepatoyctes, respectively. After 90 min exposure to 100 nM somatostatin the lumens of bile ductules have collapsed (c) while biliary canaliculi are unaffected (d). Four to 5 min after the addition of secretin (100 nM) to the somatostatin treated bile ductules (e) or hepatocytes (f). Note the rapid expansion of the bile duct lumens (arrow), and the lack of effected on the biliary canaliculi between hepatocytes (arrows). Originial magnification (×200). (JPEG 1.81 mb)

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High resolution image (PSD 8.53 mb)
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Supplementary Fig. S2

Gamma-glutamyl transpeptidase activity in co-cultured porcine hepatocytes maintained in 2% DMSO, 1% DMSO, or 0.5% DMSO. Phase contrast micrographs of hepatocytes maintained in medium supplemented with different concentrations of DMSO for 3 wk; a 2% DMSO, b 1% DMSO, or c 0.5% DMSO. Note minimal staining in cells maintained in higher concentrations of DMSO and demonstrating hepatocyte-like morphology and note intense staining at apical surfaces of the lumens of multicellular ductules formed in 0.5% DMSO (c). (JPEG 2.82 mb)

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High resolution image (PSD 14.8 mb)

Copyright information

© The Society for In Vitro Biology 2011

Authors and Affiliations

  • Thomas J. Caperna
    • 1
  • Le Ann Blomberg
    • 1
  • Wesley M. Garrett
    • 1
  • Neil C. Talbot
    • 1
  1. 1.U.S. Department of Agriculture, Agricultural Research Service, ANRIAnimal Biosciences and Biotechnology LaboratoryBeltsvilleUSA